Garcinol之癌症化學預防與神經保護活性和機制之探討

Studies on the Mechanisms of Cancer Chemopreventive and Neuroprotective Effects of Garcinol

廖瓊禾(Chiung-Ho Liao)

指導教授 ： 林仁混

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.01435

摘要

Garcinol乃自藤黃科植物如Garcinia indica、Garcinia cambogia、garciniahuillkensis等果實外殼中純化出來具 polyisoprenylated benzophenone 官能基的衍生物，Garcinia indica等乃來自印度的一種類似柑橘的果實，主要為增加色香味而添加於咖哩上或作為印度傳統醫療藥用植物，其富含多種代謝衍生之化合物，包括氧二苯甲酮、氫氧檸檬酸、黃酮類、苯甲酮類等。本篇論文主要研究藤黃類植物中的苯甲酮類化合物之生物活性，我們的研究證實Garcinol具有抗氧化、抗發炎、癌症的化學預防及保護神經細胞等功能。
過去，關於Garcinol於癌症化學預防的研究指出，此化合物具有抑制老鼠大腸瘜肉生成之功能。本篇論文中，我們首先研究Garcinol對人類大腸癌細胞的生長及轉移之影響，我們發現Garcinol抑制了癌細胞黏著相關蛋白（FAK）的磷酸化，且進一步影響其下游的訊號蛋白如Src、ERK、Akt等的活化，在癌細胞轉移方面，Garcinol亦影響MMP-7蛋白的表現，這些訊號蛋白的抑制影響了大腸癌細胞的存活與轉移；關於Garcinol誘發大腸癌細胞凋亡方面，主要受影響的蛋白有Caspase-3、PARP等，這些結果證實Garcinol可能發展為一種預防癌症之藥物。進一步地，我們研究Garcinol於抗發炎及化學癌症預防的訊號傳遞機制之影響，在發炎反應活化的老鼠巨噬細胞株上，發現其明顯抑制LPS誘發之iNOS、COX-2及TNF-a蛋白的表現，且降低了真核生物細胞中的核轉錄因子（NF-kB） 的基因調節作用，從我們的實驗結果上，發現此訊息傳遞過程中，Garcinol主要影響了核轉錄因子激活酵素（IkBa）、細胞外調節蛋白的激活酵素（ERK） 和p38細胞分裂素活化蛋白的激活酵素（MAPK），具重要的調控作用。
關於神經保護方面，主要因為Garcinol具有抗氧化能力，我們證實其能保護去氧核醣核酸不受自由基攻擊，Garcinol能清除自由基及抑制xanthine氧化酵素活性，其中自由基及過氧化物為許多神經退化或損害之疾病的成因，為了進一步了解Garcinol對神經細胞的保護能力，我們利用LPS及細胞激素刺激astrocytic 細胞產生發炎反應對神經細胞造成毒害效果，在此一神經細胞與astrocyte混合培養的環境下，Garcinol抑制NO、iNOS及COX-2的生成，提高神經細胞的存活率，且觀察到神經軸突增生的現象；因此，為了證實Garcinol對神經細胞的作用機制，我們將EGF-生成之神經幹細胞培養於Garcinol及生長因子存在之條件中，經過四天的培養下，我們觀察到神經特有蛋白的表現，如MAP-2及GFAP，利用RT-PCR實驗更證實了出現於早期神經發育之神經纖維蛋白（NFL、NFM、NFH）的mRNA的產生；根據其訊號機制的研究結果，我們發現Garcinol之存在令ERK的磷酸化現象延長，其可能調控了許多與神經發育或存活相關之核轉錄因子，如CREB之磷酸化或C/EBPb之轉位現象。經由上述實驗，證實Garcinol為有用的保護神經及促進神經發育成熟之化合物，如許多天然物粹取之化合物一般，我們研究Garcinol的之結果，不僅證實此化合物具生物活性，且令我們更清楚其作用於生物體之機制。

關鍵字

Garcinol ； 大腸癌 ； 聚噬細胞 ； 抗發炎 ； 神經保護 ； 神經外生

並列摘要

Garcinol (camboginol), a polyisoprenylated benzophenone derivative, is present in Guttiferae (Garcinia indica, Garcinia cambogia and Garcinia huillkensis). Garcinia (cv. Kokum) is used as a garnish for curry and in some of the folk medicine in India, and is a rich source of secondary metabolites including xanthones, flavanoids, benzophenones, lactones and phenolic acids. In the course of our phytochemical research on biologically active phenolic compounds, the structures of polyisoprenylated benzophenones, isolated from some guttifereous plants were characaterized. Our studies have documented the effects of garcinol, involving in Garcinia extracts, on antioxidation, anti-inflammatory, chemoprevention, neuroprotection, and promoting neurite outgrowth.
The effect of garcinol on cancer chemopreventive activity against colonic aberrant crypt foci in an animal model had been reported. Therefore, garcinol can be regarded as useful candidates for drug development including anti cancer agents. In this study, we first examined the in vitro effects of garcinol on cell invasion and growth in HT-29 human colon cancer cells. To investigating the beneficial effects of tumor prevention by garcinol, we found that garcinol decreased the dose-dependent tyrosine phosphorylation of FAK, and inhibited activation of the Src, MAPK/ERK, and PI3K/Akt signaling pathways. A decrease in the protein level of MMP-7 by garcinol also contributed to hinder the invasive ability of HT-29 cells. Furthermore, garcinol exhibited significant growth suppression due to apoptosis mediated by caspase-3 activation and PARP cleavage. Our data suggest that garcinol is a well- known cytotoxic benzophenone derivative and reduces the risk of colorectal cancer proliferation and invasion. In addition, garcinol may be potential application in chemoprevention and anti-inflammatory. In this study, the mechanisms and signal transduction pathway mediated by garcinol were further investigated. Garcinol distinctly inhibited the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and TNF-a, and consequently reduced the NF-kB-dependent transcriptional activity in LPS-activated macrophages. Based on these data, we demonstrated that inhibition of LPS-induced NF-kB activation occurred through suppressing the phosphorylation of IkBa and p38 MAPK.
To elucidate the anti-oxidative and neuroprotective properties of garcinol, we performed that garcinol protected DNA from Fenton reaction-induced breakage, and inhibits xanthine oxidase activity. To further ascertain the neuroprotective effects of garcinol in inflammatory-mediated neurotoxicity, we utilized primary neuron/astrocyte co-cultures treated with LPS or cytokine. We found that garcinol reduced iNOS expression in primary astrocytes, and enhanced neuronal survival by LPS stimulation in neuron/astrocyte co-cultures. Furthermore, garcinol added for the first 4 days increased the outgrowth of neurites in EGF-generated neurospheres. We compared the expression of neuronal proteins, microtubule-associated protein 2 (MAP-2) and glial fibrillary acidic protein (GFAP), by immunoblotting analysis and RT-PCR with primer from the neurofilaments (NFL, NFM, NFH) for the early developing neurospheres maintained in the presence of EGF and/or garcinol. Garcinol significantly enhanced the expression of these neuronal proteins. To further investigate the extracellular mechanism, our data showed an important mechanism in neurite outgrowth of garcinol-treated EGF-responsive neural precursor cells involving the duration of ERK signaling and the reduction of the withdrawal serum-dependent nuclear C/EBPb level enhanced neuronal survival. Garcinol has proven to be a potent neuroprotector and has promoted neuritogenesis in cortical progenitor cells. The progression on detail biological function investigation will help understanding the value of garcinol.

並列關鍵字

Garcinol ； colon cancer ； macrophages ； antiinflammatory ； neuroprotection ； neurite outgrowth

參考文獻

1. Adachi Y, Yamamoto H, Itoh F, Hinoda Y, Okada Y, Imai K. 1999. Contribution of matrilysin (MMP-7) to the metastatic pathway of human colorectal cancers. Gut 45: 252-258.

2. Adams JM, Cory S. 1998. The Bcl-2 Protein Family: Arbiters of Cell Survival. Science 281: 1322-1326.

3. Adler V, Schaffer A, Kim J, Dolan L, and Ronai Z. 1995. UV irradiation and heat shock mediate JNK activation via alternate pathways. J. Biol. Chem. 270: 26071-26077.

4. Ahlemeyer B, and Krieglstein J. 2003. Neuroprotective effects of Ginkgo biloba extract. Cell. Mol. Life Sci. 60: 1779-1792.

5. Aletsee C, Brors D, Palacios S, Pak K, Mullen L, Dazert S, and Ryan A F. 2002. The effects of laminin-1 on spiral ganglion neurons are dependent on the MEK/ERK signaling pathway and are partially independent of Ras. Hear Res 164: 1-11.